Antibacterial and antifungal substances biphenylyl compounds

ABSTRACT

The invention relates to the use of a substance of the general form (I) to produce an antibacterial and/or antifungal drug, wherein X is a methylene group or a carbonyl group; R 1 , R 2 , and R 3  are each selected from the group comprising hydrogen, an alkyl group having a chain length of 1-4 carbon atoms, an alkoxy group having a chain length of 1-3 carbon atoms, and a halogen; R 4  and R 5  are each selected from the group comprising hydrogen and an alkyl group having a chain length of 1-4 carbon atoms; and n=3 to 6.

The invention relates to antibacterial and antimycotic active substancesand their use for production of pharmaceutical compositions.

Despite all scientific progress bacterial infections are a constantthreat to mankind.

Today's threat is primarily due to the increase of resistance toconventional antibiotics and the low number of new drugs [Gould, I. M.,“Antibiotic resistance: the perfect storm” International Journal ofAntimicrobial Agents, 2009, 34 (3), 2-5].

Methicillin-resistant germs represent a specific problem; if aStaphylococcus aureus is methicillin-resistant (so-called MRSA) oftenresistances against antibiotics of other groups (quinolones,tetracyclines, aminoglycosides, erythromycins, sulfonamides) aredisplayed.

Therefore, the problem underlying the present invention is to providenew compounds which are effective against various bacteria and fungi,especially against those which are already showing multidrug resistanceand in this context are suitable as pharmaceutical agents.

It has been shown that basic biphenyls of the general formula

wherein X is a methylene group (CH₂) or a carbonyl group (C═O), R₁, R₂and R₃ are hydrogen, an alkyl group having a chain length of 1-4 carbonatoms, (e.g. methyl (CH₃)—, ethyl (CH₂CH₃)—, propyl (CH₂CH₂CH₃)—,isopropyl (CHCH₃CH₃)—, butyl (CH₂CH₂CH₂CH₃)—, isobutyl (CHCH₃CH₂CH₃)— ortert-butyl (CCH₃CH₃CH₃)— group), an alkoxy group having a chain lengthof 1-3 carbon atoms (e.g. a methoxy (OCH₃)—, ethoxy (OCH₂CH₃)—, propoxy(OCH₂CH₂CH₃)— or isopropoxy (OCHCH₃CH₃)— group) or a halogen (e.g.Fluorine (F), chlorine (Cl), bromine (Br) or iodine (I)),R₄ and R₅ are hydrogen or an alkyl group having a chain length of 1-4carbon atoms (e.g. methyl (CH₃)—, ethyl (CH₂CH₃)—, propyl (CH₂CH₂CH₃)—,isopropyl (CHCH₃CH₃)—, butyl (CH₂CH₂CH₂CH₃)—, isobutyl (CHCH₃CH₂CH₃)—,or tert-Butyl (CCH₃CH₃CH₃)— group) andwherein n can be n=3-6, as well as salts, hydrochlorides and tautomersof those compounds have an antimicrobial and antifungal activity alsoagainst methicillin-resistant germs. Therefore, the bisphenyls asdescribed above and illustrated in the following embodiments aresuitable for inhibiting the growth of bacteria and/or fungi.Accordingly, the present invention relates to the use of the substancesas antibacterial and/or antifungal drugs.

Table 1 shows the values of the minimum inhibitory concentration (MIC)of the substances of the present invention. For comparison, thesubstance GG20-3, which differs from the substances of the presentinvention by a shortened methyl group chain (n=2) and the knownantibiotics vancomycin and tetracycline, were also measured.

Determination of the minimum inhibitory concentration (MIC)

The minimum inhibitory concentration is the lowest concentration of thetested substance which completely inhibits the growth of each testorganism, i.e. wherein after incubation the turbidity is not measurable.The relevant determination is valid if the growth control (DMSO andmedium) a distinct turbidity is shown.

Abbreviations

Staphylococcus aureus S. a., methicillin-resistent Staphylococcus aureusMRSA, Escherichia coli E. c., Pseudomonas aeroginosa P. a.,Staphylococcus epidermis S. e., Candida albicans C. a., Enterococcushirae E. h., Aspergillus niger A. n., Aspergillus fumigatus A. f., n. b.not defined.

TABLE 1 (part 1): MIC values (μg/ml)* Substance Code S.a. MRSA E.c. P.a.S.e.

GG20-3 32 32 >64 >64 16

GG20-4 4-8 8 >64 >64 4

GG20-5 16 16 n. b. n. b. n. b.

GG28 13 (3) 18 (3) n. b. n. b. n. b. Vancomycin <2 (2) Tetracyclin 64(part 2): MIC values (μg/ml)* C. a. Substance Code (Hefe) E. h. A. n. A.f.

GG20-3 64 10 (3) n. b. n. b. n. b.

GG20-4 4 11 (5) 16 64 8

GG20-5 8 n. b. n. b. n. b.

GG 28 8 (2) n. b. n. b. n. b. *for identification of multiple testings:Representation of rounded average values of logarithmic endpoints,number of tests in brackets.

An unexpected significant increase in the efficacy can be shown in allcases in the transition of the compound GG20-3 with n=2 to compounds inwhich the chain has been extended by one or more methyl groups (n≧3).

In addition, the high efficacy of the substances of the presentinvention against various germs, in particular MRSA is observed.

A particular advantage of the compounds of the present invention istheir high solubility in water. This is a major benefit in the use ofthe substances as a drug because on the one hand, the oralbioavailability of the drug is increased, on the other hand theadministration by injection of aqueous solutions is possible. In a testtrial 100 mg of the compound GG20-4 (see Table 1) could be dissolved in10 ml of distilled water, wherein the person skilled in art knows thatpharmaceuticals are typically administered in combination withpharmaceutically acceptable excipients.

A characteristic example for a substance in accordance with the presentinvention, which is both antibacterial and antifungal active, is4′-(4-bromophenyl)-4-dimethylaminobutyrophenon-hydrochloride 1 (GG20-4)with n=3:

Another example for substances according to the present invention is thecompound 4-bromo-4′-(4-dimethylaminobutyl)biphenylhydrochloride 2(GG28). This substance is not known in the literature and also has bothantibacterial and antifungal activity.

The synthesis of the antimicrobial and antifungal active substances ofthe present invention are shown in Scheme 1 and 2. They can be producedby conventional methods. The comparative compounds with a chain lengthof n=2 can be obtained from a reaction of partially substitutedacetophenones with dimethylammonium chloride and paraformaldehyde in thesense of a Mannich reaction. Compounds with the chain length of n=3 wereobtained by a reaction of substituted 4-chloro-butyrophenon derivatswith dimethylamine in the sense of a nucleophilic substitution. Thisreaction is exemplified below for the compound4′-(4-bromophenyl)-4-dimethylaminobutyrophenonhydrochloride 1 (GG20-4):

Compounds with a chain length of n=4 were obtained analogously to thereaction with substituted 5-chloro-valerianophenon derivats withdimethylamine in the sense of a nucleophilic substitution.

The compound 2 was obtained based on the compound 1 in the sense of aWolff-Kishner-Reduction following by the Huang-Minlon-Variant:

The basic biphenyls of the present invention represent high effectiveinhibitors of the growth of bacteria and fungi. They can be easily andeconomically synthesized by standard methods with high purity.Accordingly, the present invention provides compounds for inhibiting thegrowth of bacteria and/or fungi and, consequently, drugs for thetreatment of infections of bacteria and/or fungi.

Studies of analogue compounds with a linker consisting of three, fourand five carbon atoms also without ketofunction show that theantibacterial effect is dependent on the length of the side chain.Alternatively, the replacement of the N-dimethylamino group to aN-phenylpiperazino-group leads to loss of activity. In a test experimenta MIC value for 4′-(4-bromophenyl)-4-(N-phenylpiperazino)butyrophenonehydrochloride could not be determined, since the substance did notinhibit the growth of Staphylococcus aureus also after addition ofhigher concentrations.

Pathogens in the growth phase are killed with lower concentrations asthose which are in the dormant phase. In a test experiment, the testgerms were once used in the exponential phase of their growth (18 hbefore incubation), compared to an inoculum which was harvested, andused after 48 h (quiescent period). Cells which were not as capable ofdividing, showed a 2-3 fold higher MIC as the active cells. Thus, thereare indications that quiescent phase cells are less sensitive.

As evident from the explanations and examples above, the substances ofthe present invention can also be used as laboratory reagents forinhibiting the growth of bacteria and/or fungi in cell culture, similaras described for the selection marker system neomycin andneomycin-phosphotransferase system.

The preparation in accordance with the present invention is illustratedby way of examples.

EXAMPLE 1 4′-(4-bromophenyl)-4-dimethylaminobutyrophenon-hydrochloride 1

25.7 mmol (6 g) 4-brombiphenyl, 25.7 mmol (3.6 g) 4-chloro butyrylchloride and 32.2 mmol (4.2 g) aluminiumtrichloride are weighed out in a250 mL piston before 50 mL dryed dichloromethane are added. Afterstirring overnight at room temperature, the mixture is placed on ice andthe precipitated aluminum hydroxide is brought into solution withconcentrated hydrochloric acid. Subsequently, the organic phase isseparated and extracted with water as long as the water phase shows aneutral pH value. The organic phase is than dried over Na₂SO₄, and thesolvent is filtered and removed under vacuum. The residuum isrecrystallized from a mixture of cyclohexane/ethyl acetate.

1.2 mmol (500 mg) NaI are added to 1.2 mmol (184 mg) of the resultingpure product in 5 mL ethylmethylketone and heated for 1½ h at 90° C. inan oil bath. Subsequently, the solution is removed in a vacuum, 1 mL ofdimethylamine as well as 30 mL absolute ethanol are added and heated forfurther 8 h at reflux.

After removing the solvent in the vacuum, ice is added to the residueand the precipitated raw product is aspirated. Recrystallization fromcyclohexane/ethyl acetate results 1 in a yield of 82% (377 mg); mp=224°C.

¹H-NMR Data (300 MHz, DMSO-d₆); 1.94 (m., 2H, —CH₂—), 2.8 (s, 6H,N(CH₃)₂), 3.15 (m, 4H, COCH₂—; —CH₂NH(CH₃)₂), 7.70 (m, 4H, Ar—H); 7.85(d, ³J=8.7 Hz, 2H, H-3′, H-5′), 8.04 (d, ³J=8.7 Hz; 2H, H-2′, H-6′),9.21 (bs, 1H, NH). ¹³C-NMR-Data (75 MHz, DMSO-d₆): 18.4 (C-3), 35.0(C-2), 42.0 (2×CH₃), 56.1 (C-4), 122.0 (C-4″), 126.8 (2C, C-3′, C-5′),128.6 (2C, C-2′, C-6′), 129.0 (2C, C-2″; C-6″), 131.9 (2C, C-3″, C-5″),135.5 (C-1″), 138.0 (C-1′), 143.2 (C-4′), 198.2 (CO).

EXAMPLE 2 4-bromo-4′-(4-dimethylaminobutyl) biphenylhydrochloride 2

0.02 mol (6.96 g), 1, 5 mL hydrazine monohydrate and 60 mL absoluteethanol are added in a 100 mL piston. Subsequently, it is heated at thereflux until a complete solution occurs (12 h). Afterwards, 40 mLethanol are removed under vacuum and 40 mL triglycol as well as 10 g ofpotassium hydroxide and 3 ml hydrazine monohydrate are added. Themixture is heated a further hour at 80° C. (development of gas!) beforethe temperature is up-regulated until the thermometer indicates 200 to220° C. The mixture is cooled down and the resulting precipitation isaspirated and recrystallized from ethanol. Yield 85% (5.6 g); mp=245° C.

¹H-NMR-Data (300 MHz, CDCl₃): 1.39 (m, 2H, —CH₂—), 1.62 (m, 2H, —CH₂—),2.27 (s, 6H, N(CH₃)₂), 2.36 (t, ³J=8.4 Hz, 2H, —CH₂NH(CH₃)₂), 2.55 (t,³J=8.4 Hz, 2H, Ar—CH₂—), 7.18 (d, ³J=8.7 Hz; 2H, H-2′, H-6′); 7.42 (m,4H, H-3′, H-5′, H-2″, H-6″), 7.48 (d, ³J=8.7 Hz; 2H, H-3″, H-5″).¹³C-NMR-Data (75 MHz, CDCl₃): 27.4 (C-3), 29.0 (C-2), 35.7 (C−1), 45.9(2×CH₃), 59.1 (C-4), 122.0 (C-4″), 127.7 (2C, C-3′, C-5′), 128.7 (2C,C-2′, C-6′), 130.1 (2C, C-2″; C-6″), 132.2 (2C, C-3″, C-5″), 133.7(C-4′), 135.5 (C-1″), 137.6 (C-1′).

The melting points of the synthesized substances were measured with themelting point apparatus Büchi 510 device and microhotplate Thermovar(Company Reichert). The NMR spectra were measured with thenuclearmagneticresonancespectrometer Bruker ARX 300 and the IR spectra(as KBr-Presslinge) with a Perkin-Elmer FT-IR 16 PC spectrometer. Themass spectra were measured with a device of type Hewlett-Packard 5989.Elementary analysis was perfomed in the Institute of InorganicChemistry, of the CAU Kiel using a CHNS-analyzator of the companyHekatech GmbH. Unless otherwise stated, the chemicals includingvancomycin-HCl and tetracycline-HCl at the highest purity were purchasedat the company Sigma-Aldrich GmbH.

The minimum inhibitory concentrations of the substances of the presentinventions against various infectious germs, i.a. againstmethicillin-resistant Staphylococcus aureus were determined with theBouillon-microdilutionmethod in accordance with the procedure M07-A8 ofthe Clinical and Laboratory Standards Institute (Pennsylvania, USA).

Test Organisms

(Source: German Collection of Microorganisms and Cell Cultures,Braunschweig), Enterococcus hirae ATCC 10541, Staphylococcus epidermidisATCC 12228, Staphylococcus aureus ATCC 6538, methicillin-resistantStaphylococcus aureus ATCC 33592, Bacillus cereus ATCC 11778, Candidaalbicans ATCC 10231, Aspergillus niger ATCC 16404, Aspergillus fumigatusATCC 9197.

Test Vessels

Sterile 96-well microtiterplates of plastic with rounded bottoms of thetest wells (wells; BRANDplates™, Ref. 781960).

Cultivation of Test Organisms

In sterile tubes with titled agar, bacteria with Tryptic Soy Agar(Merck, Art. No. 1.05458.05), incubation 18-24 h at 34° C., fungi withSabouraud 4% Glucose Agar (Merck 1.05438.05), incubation Candidaalbicans 18-24 h at 34° C., mold fungous 7 days at 22° C.

The long-term cultures of the test organisms are incubated at titledagar at 22° C. and each inoculated after 4 weeks on fresh medium. Afterevery fifth culture passage or ascertainment of impurities to thecorrespondent culture is discarded and newly grown from the lyophilized.

Production of Inocula

Suspensions are produced on titled agar by floating with 0.9% sterileNaCl solution. The turbidity is adjusted photometrically by dilutionaccording to the turbidity of the Mc Farland-standard 0.5. Subsequently,the bacterial suspensions are diluted in the ratio of 1:10, thePi-suspension of the fungi remains undiluted.

The number of the germs in the inocula is adjusted so that there areapproximately 5×10⁵ colony forming units per milliliter afterinoculation of the test wells.

Preparing Stock Solutions of the Antibiotics

The amount of a substance to be tested in a trial is weighed out on amicrobalance with an Eppendorf reaction vessel and dissolved in anappropiate volume of dimethyl sulfoxide so that the concentration of theresulting solution corresponds to the 21 times highest final testconcentration. The stock solution is subsequently diluted in a serial ina ratio respectively 1 ad 2 with DMSO resulting in seven differentconcentrations in the wells of a predilutionplate. In the eighth wellonly DMSO is pipetted.

Loading the Plates

In each of the 96 wells of a test plate 95 μl sterile Mueller Hinton IImedium (Cation adjusted, BBL™ Ref 212322) for bacteria and sterileSabouraud 2% glucose medium (Difco™ Ref 238230) for fungis are addedwith an eight-channel pipette (Socorex 50-200 μL). 5 μL of the solutionto be tested are pipetted in each of the test wells of the plate(8-channel pipette, Eppendorf-Research, 5-10 μL). They are transferredin this way from the pre-dilutions that the horizontal row A of thehighest concentration and to row G which shows a half reducedconcentration of the substance to be tested and in row H where only DMSOis pipetted. Subsequently, 5 μL of the inocula are pipetted in the testwells using an eight-channel pipette. In one test at least two, maximumfour rows are inoculated with the same substance and the same germs. Theconcentrations of the test substance are each 128 μg/mL to 2 μg/mL.

Evaluation and Incubation of the Microtiter Plates

The prepared plates are converted to a microtiter plate reader (Anthoshtlll) which is connected to a printer. In this device, the plates areshaken for 60 seconds with high frequency before each measurement andthe absorption of the probes is measured at the wavelength of light of590 nm.

After determination of the initial values the plates are incubated at34° C. for 16 to 20 h for bacteria and Candida albicans, and 68 to 72 hat 34° C. for mould fungis.

Following this the microtiterplates are measured again as describedabove.

Determination of the Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration is the lowest concentration of thetested substance which is able to inhibit the complete growth of theparticular test organism, i.e. where after incubation no turbidity ismeasurable. The particular determination is valid whether in the growthcontrol (DMSO and medium) a distinct turbidity is observed.

1. A substance of the general formula

for use as antibacterial and/or antifungal drug, wherein X is amethylene or a carbonyl group; R₁, R₂ and R₃ are each selected from thegroup consisting of hydrogen, an alkyl group having a chain length of1-4 carbon atoms, an alkoxy group with a chain length of 1-3 carbonatoms, and a halogen; R₄ and R₅ are each selected from the groupconsisting of hydrogen and an alkyl group with a chain length of 1-4carbon atoms; and n=3 to
 6. 2. The substance of claim 1, wherein thesubstance is available as a hydrochloride, a salt, or as a tautomer. 3.The substance of claim 1, wherein the alkyl group is a methyl (CH₃)—, anethyl (CH₂CH₃)—, a propyl (CH₂CH₂CH₃)—, an isopropyl (CHCH₃CH₃)—, abutyl (CH₂CH₂CH₂CH₃)—, an isobutyl (CHCH₃CH₂CH₃)— or a tertiary butyl(CCH₃CH₃CH₃)— group.
 4. The substance of claim 1, wherein the alkoxygroup is a methoxy (OCH₃)—, an ethoxy (OCH₂CH₃)—, a propoxy(OCH₂CH₂CH₃)— or an isopropoxy (OCHCH₃CH₃)— group.
 5. The substance ofclaim 1, wherein the halogen is fluorine (F), chlorine (Cl), bromine(Br), or iodine (I).
 6. The substance of claim 1, wherein thewater-solubility of the substance is equal to or greater than 1,000mg/l.
 7. The substance of claim 1, wherein the water-solubility of thesubstance is equal to or greater than 5,000 mg/l.
 8. The substance ofclaim 1, wherein the water-solubility of the substance is equal to orgreater than 10,000 mg/l.
 9. The substance of claim 1, wherein thesubstance is  4′(4-bromphenyl)-4-dimethylaminobutyrophenonhydrochloridewith the structural formula


10. The substance of claim 1, wherein the substance is4-brom-4′-(4-dimethylaminobutyl)biphenylhydrochloride with thestructural formula